F.-Javier Gella

Determination of total and pancreatic α-amylase in human serum with 2-chloro-4-nitrophenyl-α-D-maltotrioside as substrate

Abstract A reagent and assay conditions for the determination of the catalytic concentration of α-amylase (E.C. 3.2.1.1) in serum with 2-chloro-4-nitrophenyl-α- d -maltotrioside as substrate are described. The selected reaction mixture contains 50 mmol/l 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1 (37°C), 300 mmol/l sodium chloride, 5 mmol/l calcium chloride and 450 mmol/l potassium thiocyanate. The described method is suitable for the measurement of total as well as pancreatic α-amylase by including antibodies against the salivary isoenzyme. The method shows the absence of a lag phase period, is sensitive and precise, has a large analytical range and is free from interference by hemoglobin, bilirubin and triglycerides. Comparative studies showed good correlation with other well established methods.

A simple procedure for the routine determination of aspartate aminotransferase and alanine aminotransferase with pyridoxal phosphate

The preferred techniques for determination of the catalytic activity of aspartate aminotransferase (AST; EC 2.6.1.2) are those first described by Karmen, Wroblewski and La Due [1,2] and later improved by several Societies of Clinical Chemistry [3-51, in which oxaloacetate or pyruvate formed are enzymatically reduced to malate or lactate with concomitant oxidation of NADH. More recently, it has been found that serum aminotransferases may be undersaturated with the coenzyme pyridoxal phosphate [6-Q which should be included in the reaction mixture to improve the accuracy and the diagnostic sensitivity of the aminotransferases determination. The proposed IFCC methods for AST [9,10] and ALT [ll] saturate serum aminotransferases with pyridoxal phosphate during a lo-mm period of preincubation and the reaction is then initiated by addition of 2-oxoglutarate. The IFCC recommendations, that can be considered ‘reference methods’ [12], are inappropriate for use as routine methods in many laboratories because the use of two reagents and the long preincubation period of 10 min are serious inconveniences. We have studied a modification of the IFCC methods for AST and ALT that simplifies the procedure. The proposed procedure can use commercially available reagent kits and can be easily mechanised.

Brain pyruvate oxidation in experimental thiamin-deficiency encephalopathy

Pyrithiamine-induced thiamin deficiency has been used in rat as an experimental form of Wernicke-Korsakoff encephalopathy, a disease associated with chronic alcoholism. Although the main etiological factor is known to be the lack of thiamin, the biochemical mechanisms involved in the pathogenesis remain unclear. Thiamin-dependent enzymes were studied in brain mitochondria: alpha-ketoglutarate dehydrogenase activity exhibited 40% reduction, whereas pyruvate dehydrogenase did not change significantly. Polarographic recordings of mitochondrial respiration revealed a decreased State 3, when using pyruvate/malate, alpha-ketoglutarate or glutamine as a substrate, but the respiration rates remained unchanged with glutamate or succinate. This fall in pyruvate oxidation may be due to the impairment of alpha-ketoglutarate dehydrogenase, which follows pyruvate dehydrogenase in the metabolic pathway. A time course of lactate concentration showed dramatic increases in thalamus, mid brain, hypothalamus and colliculli, consistent with the anatomopathological findings. No increases were found before the onset of neurological symptoms.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C. Part 9: Reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) 1)

Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

Production and certification of an enzyme reference material for adenosine deaminase 1 (BCR 647)

BACKGROUND We describe the preparation of a lyophilised reference material containing purified human adenosine deaminase 1 and the certification of its catalytic concentration. METHODS The enzyme was purified from human erythrocytes. RESULTS The enzyme was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts (<0.4%) of alanine aminotransferase, aspartate aminotransferase and L-lactate dehydrogenase were detected in the purified fraction. The purified adenosine deaminase had a molar mass of 41600 g/mol and an isoelectric pH at 4.7, 4.85 and 5.0. The material was prepared by diluting the purified adenosine deaminase in a matrix containing 50 mmol/l Tris-HCl buffer pH 7.4 and 30 g/l human serum albumin; dispensing in vials and freeze-drying. The batch was homogeneous and the predicted loss of adenosine deaminase activity per year on the basis of accelerated degradation studies was 0.006% at -20 degrees C and 0.04% at 4 degrees C. The certified value for adenosine deaminase catalytic concentration in the reconstituted reference material is (2.55+/-0.09) microkat/l when measured by the method that uses adenosine as substrate and glutamate dehydrogenase as auxiliary enzyme at 37 degrees C. CONCLUSIONS The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the adenosine deaminase catalytic concentration measurements.

Creatine kinase 2 mass measurement: methods comparison and study of the matrix effect.

Five different commercial immunoassays for the measurement of creatine kinase isoenzyme 2 mass concentration were compared using human plasma samples covering a wide range of creatine kinase 2 concentrations. The immunoassays studied differ in the detection systems, in the specificity of the antibodies and in the calibrators used. Intermethod comparison by regression analysis showed differences in the results of creatine kinase 2 mass concentration. The following ratios were deduced from the obtained equations: Elecsys=1.10xImmulite=1.20xIMx=1.26xACS:180= 1.33 x Stratus. The commutability of different materials prepared by diluting purified human creatine kinase 2 in biological and synthetic matrices was studied using the different immunoassays in comparison with human plasma specimens. Almost all the materials tested were not commutable.

Production and certification of an enzyme reference material for pancreatic α-amylase (CRM 476)

Abstract We describe the preparation of a lyophilized material containing purified human pancreatic α-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic a-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified α-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/1 and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at −20° C. The certified value for α-amylase catalytic concentration in the reconstituted reference material is 555 U/l ± 11 U/l when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of α-amylase catalytic concentration measurements.

Purification of human adenosine deaminase for the preparation of a reference material

The goal was to optimise a purification procedure of adenosine deaminase from human erythrocytes for the preparation of a European Reference Material. Adenosine deaminase was purified from human erythrocytes with a specific activity of 4.46 microkat/mg of protein and a catalytic concentration of 133 microkat/l. The isolation and purification procedure involved ion-exchange chromatography (STREAMLINE DEAE), and two purine riboside affinity chromatographies. The purified enzyme exhibits a single band in SDS-PAGE with a molecular weight of 41600 g/mol, and three bands in PAGE, isoelectric focusing and two-dimensional electrophoresis with pI 4.7, 4.85 and 5.0.

Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608).

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was > 99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43,650 and 41,700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at -20 degrees C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2+/-1.8 U/L (1.12+/-0.03 microkat/L) when measured, at 30 degrees C, by the Recommended Method of the International Federation of Clinical Chemistry.

Assay of glutaminase activity by colorimetric determination of glutamate

Abstract An assay method for glutaminase is described in which the rate of release of glutamate from glutamine is followed in a second step by means of two coupled reactions involving glutamate dehydrogenase and the phenazine methosulfate-catalyzed reduction of iodonitrotetrazolium chloride by NADH. The assay is rapid, sensitive, and reproducible, and may be employed to determine glutaminase activity in crude as well as in purified enzyme preparations.